SUMMARY1. Previous work has.shown that the sodium efflux from the axons of Loligo forbesi increa 2. The increase in efflux in lithium was unaffected by ouabain but was abolished by removal of external calcium; in these respects it differed from the potassium-dependent sodium efflux which was abolished by ouabain but not reduced by removal of external calcium.3. Strontium but not magnesium could replace calcium in activating the ouabain-insensitive sodium efflux; lanthanum had an inhibitory effect.4. Replacing all the holine chloride or dextrose gave a rise in Na efflux which was abolishey ouabain but not by removal of external calcium.5. The rise in Na efflux resulting from partial replacement of NaCl by dextrose or choline chloride consisted of two components one of which was ouabain-insensitive and calcium-dependent and the other was inhibited by ouabain but calcium-insensitive.6. The ouabain-insensitive component of the Na efflux was activated by low concentrations of Na, Li or K but inhibited by high concentrations of Na and to a lesser extent Li. The inhibiting effect of high Na was of the kind expected if these ions displace calcium from an external site.7. The ouabain-insensitive component of the Na efflux was abolished by cyanide, had a Qlo of 2-7; and was roughly proportional to [Na]?. It was much more variable in magnitude than the ouabain-sensitive, potassiumdependent component of the sodium efflux. 8. The calcium influx increased five to fortyfold when external NaCl

After injury to the cell membrane, rapid resealing of the membrane occurs with little loss of intracellular contents. This process has been studied by measurement of the rate of dye loss after membrane puncture in both the sea urchin embryo and 3T3 fibroblasts. Resealing of disrupted cell membranes requires external calcium that can be antagonized by magnesium. Block of multifunctional calcium/calmodulin kinase, which regulates exocytotic vesicle availability at synapses, and of kinesin, which is required for outward-directed transport of vesicles, inhibited membrane resealing. Resealing was also inhibited by botulinum neurotoxins B and A, suggesting that the two synaptosomal-associated proteins synaptobrevin and SNAP-25 also participate in resealing. This pattern of inhibition indicates that the calcium-dependent mechanisms for cell membrane resealing may involve vesicle delivery, docking, and fusion, similar to the exocytosis of neurotransmitters.

Many metazoan cells inhabit mechanically stressful environments and, consequently, their plasma membranes are frequently disrupted. Survival requires that the cell rapidly repair or reseal the disruption. Rapid resealing is an active and complex structural modification that employs endomembrane as its primary building block, and cytoskeletal and membrane fusion proteins as its catalysts. Endomembrane is delivered to the damaged plasma membrane through exocytosis, a ubiquitous Ca2+-triggered response to disruption. Tissue and cell level architecture prevent disruptions from occurring, either by shielding cells from damaging levels of force, or, when this is not possible, by promoting safe force transmission through the plasma membrane via protein-based cables and linkages. Prevention of disruption also can be a dynamic cell or tissue level adaptation triggered when a damaging level of mechanical stress is imposed. Disease results from failure of either the preventive or resealing mechanisms.

The defective gene responsible for Duchenne muscular dystrophy in humans and the dystrophic condition in mdx mice results in a lack of dystrophin at first thought to be localized to the triads, but more recently found on the cytoplasmic side of the sarcolemma of skeletal muscle fibres. Because the total calcium content of dystrophic fibres is significantly raised, we have compared the intracellular free calcium concentration [( Ca2+]i) in skeletal muscle in normal and mdx mice. We find that [Ca2+]i is markedly elevated in mdx muscle fibres compared with normal fibres, both at rest and during stimulation. By measuring protein degradation rates and manipulating [Ca2+]i, we have been able to demonstrate directly that the elevation of [Ca2+]i in mdx fibres results in an enhanced net degradation of muscle proteins.

Although the regulation of events in the cell division cycle by calcium or other cations has been the subject of much interest and speculation, experimental studies have been hampered by the difficulty of measuring submicromolar intracellular free calcium concentrations ([Ca2+]i) over an entire cell cycle. We now describe experiments using a new fluorescent calcium chelator, fura-2 (see Fig. 1c for structure), for continuous measurement of [Ca2+]i from fertilization through the first cleavage of individual eggs of the sea urchin Lytechinus pictus. We also show for comparison the results of parthenogenetic activation by ammonia. In addition to the known transient rise of [Ca2+]i at fertilization, further peaks are now revealed during pronuclear migration, nuclear envelope breakdown, the metaphase/anaphase transition and cleavage. Parthenogenetic activation by ammonia also elicits a sustained rise starting at nuclear envelope breakdown.